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Simultaneous analyzing multiple protein and nucleic acid biomarkers in one clinical sample can provide clinically valuable results for the diagnosis, monitoring, and management of diseases. However, current diagnostic platforms primarily rely on independent detection of proteins/nucleic acids, which is time-consuming and labor-intensive. Here, we develop a Highly-Parallelized and Low-Sample-Size (HPLSS) chip for simultaneously detecting protein and nucleic acid biomarker in hepatocellular carcinoma (HCC). We assess the limit of detections for five different markers (0.14 ng mL-1 for alpha fetoprotein (AFP), 0.09 ng mL-1 for protein induced by vitamin K absence or antagonist-II (PIVKA II), 7.94 copies μL-1 for microRNA-21, 4.88 copies μL-1 for microRNA-122, and 5.83 copies μL-1 for microRNA-223). We also evaluate the performance using clinical samples and compared it with commercial kits. Our method exhibited a small sample volume required (∼20 μL), multiplexed ability and adjustable throughput (five targets in each of six samples in one assay). Importantly, the combined evaluation of multiple biomarkers of different dimensions can improve the specificity and sensitivity of diagnosis. This study has achieved the multidimensional detection of tumor biomarkers, which has great potential for early and accurate cancer diagnosis. [Display omitted] • A highly-parallelized and low-sample-size chip for multi-biomarkers detection in hepatocellular carcinoma was developed. • AFP and PIVKA II antigen, miR-21, miR-122, and miR-223 in 20 μL plasma were measured using our chip. • For 23 samples, detection results with consistency R2 ≥ 0.985 were obtained between our chip assay and RT-qPCR & ELISA. • Combined detection showed higher sensitivity, specificity, and consistency than single-/multi- detection of antigen/miRNA. [ABSTRACT FROM AUTHOR]